Han Shih

PEG hydrogels formed by thiol-ene photo-click chemistry and their effect on the formation and recovery of insulin-secreting cell spheroids

Hydrogels provide three-dimensional frameworks with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. While recent research efforts have created diverse macromer chemistry to form hydrogels, the mechanisms of hydrogel polymerization for in situ cell encapsulation remain limited. Hydrogels prepared from chain-growth photopolymerization of poly(ethylene glycol) diacrylate (PEGDA) are commonly used to encapsulate cells. However, free radical associated cell damage poses significant limitation for this gel platform. More recently, PEG hydrogels formed by thiol-ene photo-click chemistry have been developed for cell encapsulation. While both chain-growth and step-growth photopolymerizations offer spatial-temporal control over polymerization kinetics, step-growth thiol-ene hydrogels offer more diverse and preferential properties. Here, we report the superior properties of step-growth thiol-ene click hydrogels, including cytocompatibility of the reactions, improved hydrogel physical properties, and the ability for 3D culture of pancreatic β-cells. Cells encapsulated in thiol-ene hydrogels formed spherical clusters naturally and were retrieved via rapid chymotrypsin-mediated gel erosion. The recovered cell spheroids released insulin in response to glucose treatment, demonstrating the cytocompatibility of thiol-ene hydrogels and the enzymatic mechanism of cell spheroids recovery. Thiol-ene click reactions provide an attractive means to fabricate PEG hydrogels with superior gel properties for in situ cell encapsulation, as well as to generate and recover 3D cellular structures for regenerative medicine applications.

Visible‐Light‐Mediated Thiol‐Ene Hydrogelation Using Eosin‐Y as the Only Photoinitiator

The utility of visible-light-mediated polymerization in tissue engineering has been limited due to the necessary use of potentially cytotoxic coinitiator and comonomer. Here, we report a visible-light-mediated thiol-ene hydrogelation scheme using eosin-Y as the only photoinitiator. Under visible light exposure, rapid and highly tunable step-growth gelation is achieved using PEG-norbornene and a model cross-linker dithiothreitol. In addition to investigating the gelation kinetics and properties of thiol-ene hydrogels formed by this new gelation scheme, we also report high cytocompatibility of these hydrogels using human mesenchymal stem cells (hMSCs) and pancreatic MIN6 β-cells.

Gelatin hydrogels formed by orthogonal thiol–norbornene photochemistry for cell encapsulation

Covalently cross-linked gelatin hydrogels have received considerable attention in biomedical fields due to the inherent bioactivity of gelatin and the stability of covalent bonds linking the gelatin chains. Derivatives of gelatin, such as gelatin-methacrylamide (GelMA), can be cross-linked into covalent hydrogels through radical-mediated chain-growth photopolymerization. However, accumulating evidence suggests that chain-growth polymerized hydrogels may not be ideal for the encapsulation of cells and proteins prone to radical-mediated damage. The formation of heterogeneous kinetic chains following chain-growth polymerization of (meth)acrylates or (meth)acrylamides may also hinder molecular transport or alter cell-cell/cell-material interactions. This study presents a new synthesis route for preparing norbornene-functionalized gelatin (GelNB) that could be used to form orthogonally cross-linked gelatin-based hydrogels via a thiol-ene photo-click reaction. GelNB was synthesized through reacting gelatin with carbic anhydride in aqueous buffered solution, and the degree of norbornene substitution was controlled by adjusting the reaction time and the solution pH value. GelNB hydrogels were prepared by step-growth thiol-ene photopolymerization using multifunctional thiols as gel cross-linkers and the degree of GelNB hydrogel cross-linking was tuned by adjusting the thiol concentration, GelNB content, or cross-linker functionality. The cytocompatibility of orthogonally cross-linked GelNB hydrogels were demonstrated by in situ photo-encapsulation of human mesenchymal stem cells (hMSCs). When compared with the chain-growth GelMA hydrogels, the orthogonally cross-linked GelNB hydrogel promoted a faster and higher degree of cell spreading.

Photoclick Hydrogels Prepared from Functionalized Cyclodextrin and Poly(ethylene glycol) for Drug Delivery and in Situ Cell Encapsulation

Polymers or hydrogels containing modified cyclodextrin (CD) are highly useful in drug delivery applications, as CD is a cytocompatible amphiphilic molecule that can complex with a variety of hydrophobic drugs. Here, we designed modular photoclick thiol-ene hydrogels from derivatives of βCD and poly(ethylene glycol) (PEG), including βCD-allylether (βCD-AE), βCD-thiol (βCD-SH), PEG-thiol (PEGSH), and PEG-norbornene (PEGNB). Two types of CD-PEG hybrid hydrogels were prepared using radical-mediated thiol-ene photoclick reactions. Specifically, thiol-allylether hydrogels were formed by reacting multiarm PEGSH and βCD-AE, and thiol-norbornene hydrogels were formed by cross-linking βCD-SH and multiarm PEGNB. We characterized the properties of these two types of thiol-ene hydrogels, including gelation kinetics, gel fractions, hydrolytic stability, and cytocompatibility. Compared with thiol-allylether hydrogels, thiol-norbornene photoclick reaction formed hydrogels with faster gelation kinetics at equivalent macromer contents. Using curcumin, an anti-inflammatory and anticancer hydrophobic molecule, we demonstrated that CD-cross-linked PEG-based hydrogels, when compared with pure PEG-based hydrogels, afforded higher drug loading efficiency and prolonged delivery in vitro. Cytocompatibility of these CD-cross-linked hydrogels were evaluated by in situ encapsulation of radical sensitive pancreatic MIN6 β-cells. All formulations and cross-linking conditions tested were cytocompatible for cell encapsulation. Furthermore, hydrogels cross-linked by βCD-SH showed enhanced cell proliferation and insulin secretion as compared to gels cross-linked by either dithiothreitol (DTT) or βCD-AE, suggesting the profound impact of both macromer compositions and gelation chemistry on cell fate in chemically cross-linked hydrogels.